Determination techniques are widely used to analyze various enzymes or physiologically active substances contained in biological liquid samples such as human sera, based on the specificity of enzyme reactions, and then make use of the results for diagnosis of diseases and clinical examinations. In these determination techniques, there is a well known method of determining various physiologically active substances, by quantitatively converting the physiologically active substances to be detected to glucose-6-phosphate (G6P), dehydrogenating the resulting glucose-6-phosphate (G6P) in the presence of glucose-6-phosphate dehydrogenase (G6PDH) and NAD or NADP to produce 6-phosphogluconate (6PG), as shown by the following reaction formula (I): ##STR1## measuring the amount of the simultaneously produced NADH or NADPH by the absorbance thereof, and thus quantifying the physiologically active substances to be detected.
As physiologically active substances which can quantitatively be determined by converting to G6P, there may be mentioned, for example, creatine kinase (CK) which is the diagnostic indicator of cardiac infarction, muscular dystrophy, and the like; amylase (AMY) which serves as the diagnostic indicator of pancreatic disease, liver disease and the like; and glucose (Glc) which serves as the diagnostic indicator of diabetes and the like. It is possible to yield NAD(P)H by the reaction of the formula (I) from the quantitatively converted G6P, measure the increase of NAD(P)H, for example, from the change of absorbance at 340 nm, analyze the level of the physiologically active substances from the measured value, and thereby perform diagnosis of diseases and clinical examinations.
In the conventional determination systems, however, if the content of the physiologically active substances in the sample exceeded a certain amount, the amount of the NAD(P)H produced by a predetermined component ratio of a reagent used and within a predetermined reaction time would sometimes not increase quantitatively, and therefore it was not possible to accurately measure the content of the physiologically active substances. Therefore, when the initial measurement showed that a high content of the physiologically active substances was included in the sample, it was necessary to suitably dilute the sample and repeat the determination procedure.
The object of the present invention is to provide a means which enables accurate determination even in high concentration regions where accurate determination was not possible in the conventional methods.